The present invention is directed to methods of synthesizing high purity oligomeric compounds. The methods are directed to support bound syntheses where the attachment to the support is from a nucleosidic heterocyclic base moiety. More particularly the present methods provide for the preparation of higher purity oligomers having reduced levels of full length byproducts containing abasic sites.
Oligonucleotides and their analogs have been developed and used in molecular biology in a variety of procedures as probes, primers, linkers, adapters, and gene fragments. Modifications to oligonucleotides used in these procedures include labeling with nonisotopic labels, e.g. fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules. Other modifications have been made to the ribose phosphate backbone to increase the nuclease stability of the resulting analog. Examples of such modifications include incorporation of methyl phosphonate, phosphorothioate, or phosphorodithioate linkages, and 2xe2x80x2-O-methyl ribose sugar units. Further modifications include those made to modulate uptake and cellular distribution. With the success of these compounds for both diagnostic and therapeutic uses, there exists an ongoing demand for improved oligonucleotides and their analogs.
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. For disease states, classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect may be obtained with minimal side effects. It is therefore a general object of such therapeutic approaches to interfere with or otherwise modulate gene expression, which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence. Several oligonucleotides are currently undergoing clinical trials for such use. Phosphorothioate oligonucleotides are presently being used as such antisense agents in human clinical trials for various disease states, including use as antiviral agents.
Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate their action. Several recent reports describe such interactions (see Bielinska, A., et. al., Science, 1990, 250, 997-1000; and Wu, H., et. al., Gene, 1990, 89, 203-209).
In addition to such use as both indirect and direct regulators of proteins, oligonucleotides and their analogs also have found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides and their analogs to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligomeric compounds via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides and their analogs are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides and their analogs as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides and their analogs, both natural and synthetic, are employed as primers in such PCR technology.
Oligonucleotides and their analogs are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et al., Eds., Current Publications, 1993. Such uses include as synthetic oligonucleotide probes, in screening expression libraries with antibodies and oligomeric compounds, DNA sequencing, in vitro amplification of DNA by the polymerase chain reaction, and in site-directed mutagenesis of cloned DNA. See Book 2 of Molecular Cloning, A Laboratory Manual, supra. See also xe2x80x9cDNA-protein interactions and The Polymerase Chain Reactionxe2x80x9d in Vol. 2 of Current Protocols In Molecular Biology, supra.
Oligonucleotides and their analogs can be synthesized to have customized properties that can be tailored for desired uses. Thus a number of chemical modifications have been introduced into oligomers to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include those designed to increase binding to a target strand (i.e. increase their melting temperatures, Tm), to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides and their analogs, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.
The chemical literature discloses numerous well-known protocols for coupling nucleosides through phosphorous-containing covalent linkages to produce oligonucleotides of defined sequence. One of the most routinely used protocols is the phosphoramidite protocol (see, e.g., Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach, Beaucage, S. L.; Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311 and references cited therein; and The synthesis of Modified Oligonucleotides by the Phosphoramidite Approach and their applications, Beaucage, S. L.; Iyer, R. P., Tetrahedron, 1993, 49, 6123-6194 and references cited therein), wherein a nucleoside or oligonucleotide having a free hydroxyl group is reacted with a protected cyanoethyl phosphoramidite monomer in the presence of a weak acid to form a phosphite-linked structure. Oxidation of the phosphite linkage followed by hydrolysis of the cyanoethyl group yields the desired phosphodiester or phosphorothioate linkage.
Phosphoramidites are commercially available from a variety of commercial sources (included are: Glen Research, Sterling, Va.; Amersham Pharmacia Biotech Inc., Piscataway, N.J.; Cruachem Inc., Aston, Pa.; Chemgenes Corporation, Waltham, Mass.; Proligo LLC, Boulder, Colo.; PE Biosystems, Foster City Calif.; Beckman Coulter Inc., Fullerton, Calif.).
The synthesis of oligonucleotides has classically involved obtaining a desired product which was in itself a challenge. The synthesis of oligonucleotides has more recently evolved to the point that routine syntheses are being performed on kilogram scale. Moving forward the next step is the synthesis of oligonucleotides and analogs on ton scale. The evolution of oligonucleotide synthetic techniques toward large scale is requiring a reevaluation of each aspect of the synthetic process.
One such aspect is the site of attachment of the growing oligonucleotide to a support media. The site of attachment of the first nucleoside to the solid support can have profound affects on certain impurities that are routinely found to be present in the purified oligomeric compounds ultimately isolated. During standard coupling reactions a growing oligomer is repeatedly subjected to a variety of reaction conditions. For example standard phosphoramidite synthesis conditions include deprotection of the 5xe2x80x2-hydroxyl group, activation of the phosphoramidite, coupling of the activated phosphoramidite to the 5xe2x80x2-hydroxyl, capping of unreacted sites and oxidation of the phosphite to a phosphotriester. Repeatedly subjecting the growing oligomer to these reactions to effect further couplings is known to cause a small degree of unwanted side reactions to occur. Certain of these are essentially transparent in that they don""t lead to byproducts that will show up as impurities in the final HPLC purified oligomer. These byproducts are either removed during the wash cycles or during purification.
Oligomeric compounds are being routinely prepared on large scale for pharmaceutical use which requires high purity. These compounds are subjected to rigorous standards for purification and analysis prior to being used in for example human clinical trials. Certain byproducts are found to survive even the HPLC purification process and are very difficult to remove from the desired product. The reason for their persistence is that they are so structurally similar to the final desired product they aren""t removed by standard HPLC. An especially difficult byproduct results from the cleavage of the glycosyl bond of one or more nucleosides in the oligomer. If a single glycosyl bond is cleaved creating an abasic site the resulting byproduct is identical to the final compound except for that one heterocyclic base moiety that was cleaved. This is one of the most difficult byproducts to detect and remove from the final product.
An inherent problem encountered using the support bound phosphoramidite method of oligomer synthesis is an abasic site in the final product caused by depurination of one or more of the linked nucleosides. It has been shown that certain sites are more prone e.g. more labile under these conditions to depurination than others including the 3xe2x80x2-terminal nucleoside and nucleosides close to the 3xe2x80x2-terminus. Depurination is more likely for nucleosides near the 3xe2x80x2-terminal because these sites are subjected to more coupling cycles. An especially labile position is a 3xe2x80x2-terminal purine nucleoside during treatment with an acidic reagent to remove the 5xe2x80x2-hydroxyl group. This lability is enhanced relative to other positions due to the electronic effects of the 5xe2x80x2- and 3xe2x80x2-substituents. The presence of an abasic site resulting from depurination or other reasons in an oligomeric compound is further known to enhance xcex2-elimination (Toshinori et al., Nucleic Acids Research, 1994, 22, 4997-5003). Current attempts to reduce this risk of a 3xe2x80x2-abasic site in the final oligomer has been to alter the conditions of deprotecting the 5xe2x80x2-hydroxyl group during the first coupling.
One reaction condition that has been modified and studied with respect to depurination is the acid deprotection step. Studies have shown that the rate of depurination is decreased with lesser concentrations of weaker acids such as dichloroacetic acid as opposed to the industry standard of trichloroacetic acid (Septak, Michael, Nucleic Acids Research, 1996, 24, 3053-3058). Alternative nucleobase protecting groups have been used during the oligomerization process to reduce depurination (McBride et al., J. Am. Chem. Soc., 1986, 108, 2040-2048; McBride, et al., Tetrahedron Letters, The presence of a abasic site in an oligomeric compound is further known to enhance xcex2-elimination (Toshinori et al., Nucleic Acids Research, 1994, 22, 4997-5003). 1983, 24, 2953-2956).
There currently exists a need in the art for methods of synthesizing oligomeric compounds that reduce or eliminate byproducts that are not removed during standard purification.
Solid phase methods for the preparation of deoxyribooligonucleotides having 3xe2x80x2-peptide conjugates using a deoxycytidine attached to a solid support via the base are disclosed in Napoli et al., Bioorganic and Medicinal Chemistry, 1999, 7, 395-400.
Solid phase triester methodologies have been used to prepare cyclic oligodeoxynucleotides where the first deoxynucleoside attached to a polydimethylacrylamide support is attached at the base (Barbato et al., Tetrahedron, 1989, 45, 4523-4536; and Barbato et al., Tetrahedron Letters, 1987, 28, 5727-5728).
Attachment of a heterocyclic base to a solid support for oligomer synthesis using a linkage to the base has been previously reported (Waldvogel et al., Helvetica Chimica Acta, 1998, 81, 46-58). In a representative example an amino protecting group is used to protect the exocyclic amino functionality of a purine which is itself attached to a phosphate blocking group. The oligomerization proceeds from a purine not a nucleoside attached to a solid support.
The present invention is directed to methods for preparation of oligomers using support bound processes wherein the attachment of the growing oligomer to the support medium is through the heterocyclic base moiety of a base forming the oligomeric.
In one embodiment, methods are provided for preparing an oligomeric compound having at least one moiety of formula: 
wherein:
Q is an internucleoside linkage;
Bx is an optionally blocked heterocyclic base moiety, for example, adenine, N6-benzoyladenine, 2-aminoadenine, cytosine, Nxe2x80x2-benzoylcytosine, 5-methylcytosine, N4-benzoyl-5-methylcytosine, thymine, uracil, guanine or N isobutvrylguanine;
Bxx is a purine or purine analog;
each R is, independently, hydrogen or an optionally protected substituent group;
L is a bifunctional linking moiety, preferably moiety that attaches the support medium to the oligomeric compound at a heterocyclic base functional group; and
SM is a support medium;
comprising the steps of:
(a) providing a compound of formula: 
xe2x80x83wherein:
T1 is a 5xe2x80x2-hydroxyl protecting group; and
T2 is a hydroxyl blocking group, a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide or a conjugate group, and is preferably, a hydroxyl blocking group, for example, xe2x80x94C(xe2x95x90O)Rd, wherein Rd is C1 to C12 alkyl, such as CH3, and in some preferred embodiments is base labile;
(b) deprotecting the 5xe2x80x2-hydroxyl protecting group to form a deprotected hydroxyl group;
(c) treating the deprotected hydroxyl group with a further compound having the formula: 
xe2x80x83wherein:
T3 is a 5xe2x80x2-hydroxyl protecting group, a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide or a conjugate group; and
T4 is a reactive PIII species for forming an internucleoside linkage;
and an activating agent, for example, 1-H-tetrazole, for a time and under conditions effective to form an extended oligomeric compound;
d) treating the extended oligomeric compound with a capping agent to form a capped compound;
e) treating the capped compound with an oxidizing agent, for example, oxaziridine, preferably, 10-camphorsulphonyl oxazaridine, 2-phenylsulphonyl-3-phenyl oxazaridine, 2-(phenyl sulphonyl)-3-(3-nitrophenyl)oxazaridine, or 8,8-dihalo-10-camphorsulphonyl oxazaridine; and
f) optionally repeating steps b through e one or more additional cycles to form the oligomeric compound.
In some preferred embodiments, the oligomeric compound is further treated with a reagent effective to form a deblocked oligomeric compound, for example, a basic solution such as concentrated ammonium hydroxide. In some preferred embodiments, the reagent is effective to cleave the oligomeric compound from the support medium.
In other preferred embodiments, the deblocked oligomeric compound is further treated with a reagent effective to cleave the oligomeric compound from the support medium.
In preferred embodiments of the present invention, each substituent group that is defined by R is, independently, hydroxyl, C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, halogen, amino, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, O-alkenyl, O-alkynyl, S-alkyl, S-alkenyl, S-alkynyl, NH-alkyl, NH-alkenyl, NH-alkynyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, or polyether;
or each substituent group has one of formula I or II: 
xe2x80x83wherein:
Z0 is O, S or NH;
J is a single bond, O or C(xe2x95x90O);
E is C1-C10 alkyl, N(R1)(R2), N(R1)(R5), Nxe2x95x90C(R1)(R2), Nxe2x95x90C(R1)(R5) or has one of formula III or IV; 
each R6, R7, R8, R9 and R10 is, independently, hydrogen, C(O)R11, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;
or optionally, R7 and R8, together form a phthalimido moiety with the nitrogen atom to which they are attached;
or optionally, R9 and R10, together form a phthalimido moiety with the nitrogen atom to which they are attached;
each R11, is, independently, substituted or unsubstituted C1-C10 alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;
R5 is Txe2x80x94L1,
T is a bond or a linking moiety;
L1 is a chemical functional group, a conjugate group or a solid support material;
each R1 and R2 is, independently, H, a nitrogen protecting group, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, wherein said substitution is OR3, SR3, NH3+, N(R3)(R4) guanidino or acyl where said acyl is an acid amide or an ester;
or R1 and R2, together, are a nitrogen protecting group or are joined in a ring structure that optionally includes an additional heteroatom selected from N and O;
or R1, T and L1, together, are a chemical functional group, for example, a primary or secondary amino group;
each R3 and R4 is, independently, H, C1-C10 alkyl, a nitrogen protecting group, or R3 and R4, together, are a nitrogen protecting group;
or R3 and R4 are joined in a ring structure that optionally includes an additional heteroatom selected from N and O;
Z4 is OX, SX, or N(X)2;
each X is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(xe2x95x90NH)N(H)R5, C(xe2x95x90O)N(H)R5 or OC(xe2x95x90O)N(H)R5;
R5 is H or C1-C8 alkyl;
Z1, Z2 and Z3 comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;
Z5 is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R1)(R2) OR1, halo, SR1 or CN;
each q1 is, independently, an integer from 1 to 10;
each q2 is, independently, 0 or 1;
q3 is 0 or an integer from 1 to 10;
q4 is an integer from 1 to 10;
q5 is from 0, 1 or 2; and provided that when q3 is 0, q4 is greater than 1.
In some preferred embodiments, the oligomeric compound comprises from about 5 to about 50 monomer subunits, preferably 10 to about 30 monomer subunits, and more preferably comprises from about 15 to 25 monomer subunits.
In particularly preferred embodiments of the present invention oligomeric compounds of the following formula are prepared: 
wherein:
mm is from about 5 to 50, preferably from about 10 to 30 and more preferably, from about 15 to 25;
T5 is H, a hydroxyl blocking group, a nucleoside, an oligonucleoside, a nucleotide an oligonucleotide or a conjugate group;
T6 is H, a hydroxyl protecting group, a nucleoside, an oligonucleoside, a nucleotide an oligonucleotide or a conjugate group;
each Xa is, independently, O or S;
each Xb is, independently, OH, SH or NRaRb;
each Ra and Rb is, independently, H, a nitrogen protecting group, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, wherein said substitution is hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl or alkynyl;
wherein T1, Bxx, Bx, R, L, and SM are defined as above; comprising the steps of:
(a) providing a compound of the formula: 
(b) deprotecting said 5xe2x80x2-hydroxyl protecting group to form a deprotected hydroxyl group;
(c) treating said deprotected hydroxyl group with a further compound having the formula: 
xe2x80x83wherein:
T4 is a phosphoramidite;
and an activating agent for a time and under conditions effective to form an extended oligomeric compound;
d) treating said extended oligomeric compound with a capping agent to form a capped compound;
e) treating said capped compound with an; and
f) optionally repeating steps b through e one or more times to form said oligomeric compound.